The Experts below are selected from a list of 1476 Experts worldwide ranked by ideXlab platform
Carla Denise Bonan - One of the best experts on this subject based on the ideXlab platform.
-
Investigation into effects of antipsychotics on Ectonucleotidase and adenosine deaminase in zebrafish brain
Fish Physiology and Biochemistry, 2015Co-Authors: Kelly Juliana Seibt, Mauricio Reis Bogo, Renata Oliveira, Mario Roberto Senger, Carla Denise BonanAbstract:Antipsychotic agents are used for the treatment of psychotic symptoms in patients with several brain disorders, such as schizophrenia. Atypical and typical antipsychotics differ regarding their clinical and side-effects profile. Haloperidol is a representative typical antipsychotic drug and has potent dopamine receptor antagonistic functions; however, atypical antipsychotics have been developed and characterized an important advance in the treatment of schizophrenia and other psychotic disorders. Purine nucleotides and nucleosides, such as ATP and adenosine, constitute a ubiquitous class of extracellular signaling molecules crucial for normal functioning of the nervous system. Indirect findings suggest that changes in the purinergic system, more specifically in adenosinergic activity, could be involved in the pathophysiology of schizophrenia. We investigated the effects of typical and atypical antipsychotics on Ectonucleotidase and adenosine deaminase (ADA) activities, followed by an analysis of gene expression patterns in zebrafish brain. Haloperidol treatment (9 µM) was able to decrease ATP hydrolysis (35 %), whereas there were no changes in hydrolysis of ADP and AMP in brain membranes after antipsychotic exposure. Adenosine deamination in membrane fractions was inhibited (38 %) after haloperidol treatment when compared to the control; however, no changes were observed in ADA soluble fractions after haloperidol exposure. Sulpiride (250 µM) and olanzapine (100 µM) did not alter Ectonucleotidase and ADA activities. Haloperidol also led to a decrease in entpd2_mq , entpd3 and adal mRNA transcripts. These findings demonstrate that haloperidol is an inhibitor of NTPDase and ADA activities in zebrafish brain, suggesting that purinergic signaling may also be a target of pharmacological effects promoted by this drug.
-
long term proline exposure alters nucleotide catabolism and Ectonucleotidase gene expression in zebrafish brain
Metabolic Brain Disease, 2012Co-Authors: Luiz Eduardo Baggio Savio, Fernanda Cenci Vuaden, Mauricio Reis Bogo, Carla Denise Bonan, Denis B Rosemberg, Angela T S WyseAbstract:Hyperprolinemia is an inherited disorder of proline metabolism and hyperprolinemic patients can present neurological manifestations, such as seizures cognitive dysfunctions, and psychotic disorders. However, the underlying mechanisms of these symptoms are still unclear. Since adenine nucleotides play crucial roles in neurotransmission and neuromodulation, we evaluated the in vivo and in vitro effects of proline on Ectonucleotidase activities and gene expression in zebrafish brain. For the in vivo studies, animals were exposed at two proline concentrations (1.5 and 3.0 mM) during 1 h or 7 days (short- or long-term treatments, respectively). For the in vitro assays, different proline concentrations (ranging from 3.0 to 1000 μM) were tested. Short-term proline exposure did not promote significant changes on the Ectonucleotidase activities and gene expression. Long-term proline exposure significantly increased ATP catabolism in both concentrations tested (14 % and 22 %, respectively), whereas ADP and AMP hydrolysis were increased only at 3.0 mM proline (21 % and 17 %, respectively) when compared to control. Moreover, the relative gene expression of enpd3 increased in both treated groups after long-term proline, whereas enptd1 increased only at 3.0 mM proline. Proline in vitro did not promote significant changes on Ectonucleotidase activities. Altogether, these data indicate that the enzymes responsible for the control of extracellular nucleotides levels might be altered after proline exposure in zebrafish, contributing to better understand the pathophysiology of this disease. Moreover, such findings might facilitate the use of the zebrafish as a complementary vertebrate model for studying inborn errors of amino acid metabolism.
-
chronic mild hyperhomocysteinemia alters Ectonucleotidase activities and gene expression of ecto 5 nucleotidase cd73 in rat lymphocytes
Molecular and Cellular Biochemistry, 2012Co-Authors: Emilene B S Scherer, Fernanda Cenci Vuaden, Mauricio Reis Bogo, Carla Denise Bonan, Luiz Eduardo Baggio Savio, Andrea G K Ferreira, Angela T S WyseAbstract:Since mild hyperhomocysteinemia is a risk factor for cardiovascular and cerebral diseases and extracellular nucleotides/nucleosides, which are controlled by the enzymatic action of Ectonucleotidases, can induce an immune response, in the present study, we investigated the effect of chronic mild hyperhomocysteinemia on Ectonucleotidase activities and expression in lymphocytes from mesenteric lymph nodes and serum of adult rats. For the chronic chemically induced mild hyperhomocysteinemia, Hcy (0.03 μmol/g of body weight) or saline (control) were administered subcutaneously from the 30th to the 60th day of life. Results showed that homocysteine significantly decreased ATP, ADP, and AMP hydrolysis in lymphocytes of adult rats. E-NTPDases transcriptions were not affected, while the ecto-5′-nucleotidase transcription was significantly decreased in mesenteric lymph nodes of hyperhomocysteinemic rats. ATP, ADP, and AMP hydrolysis were not affected by homocysteine in rat serum. Our findings suggest that Hcy in levels similar to considered risk factor to development of vascular diseases modulates the Ectonucleotidases, which could lead to a pro-inflammatory status.
-
profile of nucleotide catabolism and Ectonucleotidase expression from the hippocampi of neonatal rats after caffeine exposure
Neurochemical Research, 2012Co-Authors: R S Da Silva, Mauricio Reis Bogo, Stefânia Konrad Richetti, Elisa Marquezan Tonial, Carla Denise BonanAbstract:Nucleotides and nucleosides play an important role in neurodevelopment acting through specific receptors. Ectonucleotidases are the major enzymes involved in controlling the availability of purinergic receptors ligands. ATP is co-released with several neurotransmitters and is the most important source of extracellular adenosine by catabolism exerted by Ectonucleotidases. The main Ectonucleotidases are named NTPDases (1–8) and 5′-nucleotidase. Adenosine is a powerful modulator of neurotransmitter release. Caffeine blocks adenosine receptor activity as well as adenosine-mediated neuromodulation. Considering the susceptibility of the immature brain to caffeine and the need for correct purinergic signaling during fetal development, we have analyzed the effects of caffeine exposure during gestational and lactational periods on nucleotide degradation and Ectonucleotidase expression from the hippocampi of 7-, 14- and 21-days-old rats. Nucleotides hydrolysis was assessed by colorimetric determination of inorganic phosphate released. Ectonucleotidases expression was performed by RT-PCR. ATP and ADP hydrolysis displayed parallel age-dependent decreases in both control and caffeine-treated groups. AMP hydrolysis increased with caffeine treatment in 7-days-old rats (75%); although there was no significant difference in AMP hydrolysis between control (non caffeine-treated) rats and 14- or 21-days caffeine-treated rats. ADP hydrolysis was not affected by caffeine treatment. Caffeine treatment in 7- and 14-days-old rats decreased ATP hydrolysis when compared to the control group (19% and 60% decrease, respectively), but 21-days-treated rats showed an increase in ATP hydrolysis (39%). Expression levels of NTPDase 1 and 5 decreased in hippocampi of caffeine-treated rats. The expression of 5′-nucleotidase was not affected after caffeine exposure. The changes observed in nucleotide hydrolysis and Ectonucleotidases expression could promote subtle effects on normal neural development considering the neuromodulatory role of adenosine.
-
chronic ethanol treatment alters purine nucleotide hydrolysis and nucleotidase gene expression pattern in zebrafish brain
Neurotoxicology, 2011Co-Authors: Eduardo Pacheco Rico, Mauricio Reis Bogo, Carla Denise Bonan, Denis B Rosemberg, Andrei Silveira Langoni, Andre Arigony Souto, Renato Dutra Dias, Diogo O SouzaAbstract:Ethanol is a widely consumed drug that acts on the central nervous system (CNS), modifying several signal transduction pathways activated by hormones and neurotransmitters. The zebrafish is an experimental model for the study of human diseases and the use of this species in biochemical and behavioral studies on alcoholism and alcohol-dependence has increased recently. However, there are no data concerning the effects of chronic ethanol exposure on the purinergic system, where extracellular nucleotides act as signaling molecules. Purinergic signaling is controlled by a group of enzymes named Ectonucleotidases, which include NTPDases and ecto-5'-nucleotidase already characterized in zebrafish brain. The aim of this study was to evaluate nucleotide hydrolysis by NTPDases and ecto-5'-nucleotidase after long-term ethanol exposure. Additionally, the gene expression patterns of NTPDases1-3 and 5'-nucleotidase were determined. Animals were exposed to 0.5% ethanol for 7, 14, and 28 days. There were no significant changes in ATP and GTP hydrolysis after all treatments. However, a decrease in ADP (46% and 34%) and GDP (48% and 36%) hydrolysis was verified after 7 and 14 days, respectively. After 7 and 14 days of ethanol exposure, a significant decrease in AMP hydrolysis (48% and 36%) was also observed, whereas GMP hydrolysis was inhibited only after 7 days (46%). NTPDase2_mv and NTPDase3 mRNA transcript levels decreased after 7 and 14 days, respectively. In contrast, ethanol increased NTPDase1, NTPDase2_mq, and NTPDase3 transcript levels after 28 days of exposure. NTPDase2_mg and 5'-nucleotidase gene expression was not altered. Therefore, the Ectonucleotidase pathway may be a target of chronic ethanol toxicity and the regulation of purinergic system could play a key role in the neurochemical mechanisms underlying the effects of ethanol on the CNS.
João José Freitas Sarkis - One of the best experts on this subject based on the ideXlab platform.
-
influence of antidepressant drugs on ecto nucleotide pyrophosphatase phosphodiesterases e npps from salivary glands of rats
Archives of Oral Biology, 2009Co-Authors: Sandra Liana Henz, Giana De Paula Cognato, Fernanda Cenci Vuaden, Mauricio Reis Bogo, Carla Denise Bonan, João José Freitas SarkisAbstract:Abstract Xerostomia is commonly caused by antidepressant drugs and ATP can influence the saliva production. Adenosine is the product of extracellular hydrolysis of adenine nucleotides in submandibular gland cells, which occurs by the action of Ectonucleotidases. In this study, we have evaluated the effect of three different antidepressants in ecto-nucleotide pyrophosphatase/phosphodiesterase (E-NPP1–3) activities in cultured cells from salivary glands. Rats received imipramine (10 mg/ml), fluoxetine (20 mg/ml) or moclobemide (30 mg/ml) by oral gavage. The drugs were administered once a day for 14 days. Our results have shown that the hydrolysis of p -nitrophenyl-5′-thymidine monophosphate increased in all treatments. These effects were not consequence of transcriptional control of E-NPP1–3 genes. The results reported here can highlight the importance of Ectonucleotidases in the most common side effect caused by antidepressant therapy.
-
intrastriatal injection of hypoxanthine alters striatal Ectonucleotidase activities a time dependent effect
Brain Research, 2008Co-Authors: Caren Serra Bavaresco, Giana De Paula Cognato, João José Freitas Sarkis, Mauricio Reis Bogo, Carla Denise Bonan, Denise Barbosa Ramos, Fabria Chiarani, Janaina Kolling, Carlos Alexandre Netto, Angela T S WyseAbstract:Abstract The aim of this study was to investigate the effects of intrastriatal injection of hypoxanthine on Ectonucleotidase (E-NTPDases and ecto-5′-nucleotidase) activities and expressions in the striatum of rats. The effect of pre-treatment with vitamins E and C on the effects elicited by this oxypurine on enzymatic activities and on thiobarbituric reactive substances (TBARS) was also investigated. The effect of pre-incubation with hypoxanthine on nucleotide hydrolysis in striatum homogenate was also determined. Adult Wistar rats were divided into (1) control and (2) hypoxanthine-injected groups. For Ectonucleotidase activity determination, the animals were sacrificed at 30 min, 24 h and 7 days after drug infusion. For the evaluation of the expression of NTPDase 1–3 and also ecto-5′-nucleotidase, TBARS assay and the influence of the pre-treatment with vitamins on Ectonucleotidase activities, the animals were sacrificed 24 h after hypoxanthine infusion. Results show that hypoxanthine infusion significantly inhibited Ectonucleotidase activities and increased TBARS only 24 h after administration. Pre-treatment with vitamins was able to prevent these effects. Moreover, ecto-5′-nucleotidase expression was increased (80%) at 24 h after hypoxanthine infusion. We suggest that these hypoxanthine-induced biochemical modifications could, at least in part, participate in the pathophysiology of Lesch Nyhan disease.
-
Ectonucleotidase activities are altered in serum and platelets of l name treated rats
Blood Cells Molecules and Diseases, 2008Co-Authors: Cristina Ribas Furstenau, Danielle Da Silva Trentin, Agnes Nogueira Gossenheimer, Denise Barbosa Ramos, Emerson Andre Casali, Maria Luiza Morais Barretochaves, João José Freitas SarkisAbstract:It is well known that hypertension is closely associated to the development of vascular diseases and that the inhibition of nitric oxide biosynthesis by administration of Nomega-Nitro-L-arginine methyl ester hydrochloride(L-NAME) leads to arterial hypertension. In the vascular system, extracellular purines mediate several effects;thus, ADP is the most important platelet agonist and recruiting ag ent, while adenosine, an end product of nucleotide metabolism, is a vasodilator and inhibitor of platelet activation and recruitment. Members of several families of enzymes, known as Ectonucleotidases, including E-NTPDases (ecto-nucleoside triphosphate diphosphohydrolase), E-NPP (ecto-nucleotide pyrophosphatase/phosphodiesterase) and 5'-nucleotidase are able to hydrolyze extracellular nucleotides until their respective nucleosides. We investigated the Ectonucleotidase activities of serum and platelets from rats made hypertensive by oral administration of L-NAME (30 mg/kg/day for 14 days or 30 mg/kg/day for 14 days plus 7 days of L-NAME washout, in the drinking water) in comparison to normotensive control rats. L-NAME promoted a significant rise in systolic blood pressure from 112 +/- 9.8 to 158 +/- 23 mmHg. The left ventricle weight index (LVWI) was increased in rats treated with L-NAME for 14 days when compared to control animals. In serum samples, ATP, ADP and AMP hydrolysis were reduced by about 27%, 36% and 27%, respectively. In platelets, the decrease in ATP, ADP and AMP hydrolysis was approximately 27%, 24% and 32%, respectively. All parameters recovered after 7 days of L-NAME washout. HPLC demonstrated a reduction in ADP, AMP and hypoxanthine levels by about 64%, 69% and 87%,respectively. In this study, we showed that Ectonucleotidase activities are decreased in serum and platelets from L-NAME-treated rats, which should represent an additional risk for the development of hypertension. The modulation of Ectonucleotidase activities may represent an approach to antihypertensive therapy via inhibition of spontaneous platelet activation and recruitment, as well as thrombus formation.
-
ontogenetic profile of Ectonucleotidase activities from brain synaptosomes of pilocarpine treated rats
International Journal of Developmental Neuroscience, 2005Co-Authors: Giana De Paula Cognato, Fernanda Cenci Vuaden, João José Freitas Sarkis, Alessandra Nejar Bruno, Carla Denise BonanAbstract:Adenosine, a well-known neuromodulator, can act as an endogenous anticonvulsant via the activation of adenosine A1 receptors. This adenine nucleoside can be produced in the synaptic cleft by the Ectonucleotidase cascade, which includes the nucleoside triphosphate diphosphohydrolase (NTPDase) family and ecto-5'-nucleotidase. It has been previously reported that Ectonucleotidase activities are increased in female adult rats submitted to the pilocarpine model of epilepsy. Several studies have suggested that the immature brain is less vulnerable to morphologic and physiologic alterations after status epilepticus (SE). Here, we evaluate the Ectonucleotidase activities of synaptosomes from the hippocampus and cerebral cortex of male and female rats at different ages (7-9, 14-16 and 27-30-day old) submitted to the pilocarpine model of epilepsy. Our results show that ATP and ADP hydrolysis in the hippocampus and cerebral cortex were not altered by the pilocarpine treatment in female and male rats at 7-9, 14-16 and 27-30 days. There were no changes in AMP hydrolysis in female and male rats submitted to the model at different ages, but a significant increase in AMP hydrolysis (71%) was observed in synaptosomes from the cerebral cortex of male rats at 27-30 days. Pilocarpine-treated male rats (60-70-day old) presented an enhancement in Ectonucleotidase activities in the synaptosomes of the cerebral cortex (33, 40 and 64% for ATP, ADP and AMP hydrolysis, respectively) and hippocampus (55, 98 and 101% for ATP, ADP and AMP hydrolysis, respectively). These findings highlight differences between the purinergic system of young and adult rats submitted to the pilocarpine model of epilepsy.
-
effects of steroid hormones on synaptosomal Ectonucleotidase activities from hippocampus and cortex of adult female rats
General and Comparative Endocrinology, 2005Co-Authors: Barbara Rucker, Ana Maria Oliveira Battastini, Cristina Ribas Furstenau, Maria Luiza Morais Barretochaves, Daniela Pochmann, Marcela Sorelli Carneiroramos, João José Freitas SarkisAbstract:Abstract Over the last few years, the effects of steroid hormones on the brain have been intensively discussed. It has been demonstrated that ATP (acting as a neurotransmitter) is hydrolyzed to adenosine in the synaptic cleft by the conjugated action of Ectonucleotidases, which include an enzyme of the E-NTPDase family (NTPDase3, apyrase, EC 3.6.1.5) and a 5′-nucleotidase (EC 3.1.3.5). The 5′-nucleotidase enzyme is able to hydrolyze AMP as well as other monophosphate nucleotides. The importance of this enzyme in the central nervous system is to participate in the adenosine formation, a nucleoside with neuroprotective properties and modulatory effects. However, several questions have been raised about the mechanisms of steroid hormones and the possible neuroprotective effects of estrogen. Thus, we examined the effects of gonadal steroid hormone deprivation, induced by ovary removal (OVX) and estradiol replacement therapy, on the Ectonucleotidase activities in synaptosomes from hippocampus and cerebral cortex of adult rats. ATP and ADP hydrolysis in synaptosomes from cerebral cortex and hippocampus did not change as a function of OVX and results demonstrated an increase in AMP hydrolysis (82%) in the animals submitted to OVX in cerebral cortex, but not in hippocampus, when compared to control and sham-operated groups. Estradiol replacement therapy reversed this effect. RT-PCR analysis showed that the enhancement of enzyme activity in cerebral cortex could be explained by the higher expression of 5′-nucleotidase, following OVX. The hormones 17β-estradiol (cyclodextrin-encapsulated 17β-estradiol), DHEAS, and pregnenolone (1.0, 2.5, and 5.0 μM) did not alter the nucleotide hydrolysis, in vitro, in synaptosomes from cortex and hippocampus of female adult rats. Results presented, herein, should be considered relevant for hormone replacement therapy, since much controversy exists surrounding this area and the relationship between adenosine and sex steroids is still poorly understood.
Simon C. Robson - One of the best experts on this subject based on the ideXlab platform.
-
dysregulation of adenosinergic signaling in systemic and organ specific autoimmunity
International Journal of Molecular Sciences, 2019Co-Authors: Marta Vuerich, Rasika P. Harshe, Simon C. Robson, Maria Serena LonghiAbstract:Exact causes for autoimmune diseases remain unclear and no cures are available. Breakdown of immunotolerance could set the stage for unfettered immune responses that target self-antigens. Impaired regulatory immune mechanisms could have permissive roles in autoreactivity. Abnormal regulatory immune cell function, therefore, might be a major determinant of the pathogenesis of autoimmune disease. All current treatments are associated with some level of clinical toxicity. Treatment to specifically target dysregulated immunity in these diseases would be a great advance. Extracellular adenosine is a signaling mediator that suppresses inflammation through activation of P1 receptors, most active under pathological conditions. Mounting evidence has linked alterations in the generation of adenosine from extracellular nucleotides by Ectonucleotidases, and associated perturbations in purinergic signaling, to the immunological disruption and loss of immunotolerance in autoimmunity. Targeted modulation of the purinergic signaling by either targeting Ectonucleotidases or modulating P1 purinergic receptors could therefore restore the balance between autoreactive immune responses; and thereby allow reestablishment of immunotolerance. We review the roles of CD39 and CD73 ectoenzymes in inflammatory states and with the dysregulation of P1 receptor signaling in systemic and organ-specific autoimmunity. Correction of such perturbations could be exploited in potential therapeutic applications.
-
expression of Ectonucleotidases in the prosencephalon of melatonin proficient c3h and melatonin deficient c57bl mice spatial distribution and time dependent changes
Cell and Tissue Research, 2015Co-Authors: Moran Homola, Simon C. Robson, Herbert Zimmermann, Martina Pfeffer, Claudia Fischer, Horstwerner KorfAbstract:Extracellular purines (ATP, ADP, AMP and adenosine) are important signaling molecules in the CNS. Levels of extracellular purines are regulated by enzymes located at the cell surface referred to as Ectonucleotidases. Time-dependent changes in their expression could profoundly influence the availability of extracellular purines and thereby purinergic signaling. Using radioactive in situ hybridization, we analyzed the mRNA distribution of the enzymes NTPDase1, -2 and -3 and ecto-5′-nucleotidase in the prosencephalon of two mouse strains: melatonin-proficient C3H and melatonin-deficient C57Bl. The mRNAs of these enzymes were localized to specific brain regions, such as hippocampus, striatum, medial habenula and ventromedial hypothalamus. NTPDase3 expression was more widely distributed than previously thought. All Ectonucleotidases investigated revealed a prominent time-dependent expression pattern. In C3H, the mRNA expression of all four enzymes gradually increased during the day and peaked during the night. In contrast, in C57Bl, ecto-5′-nucleotidase expression peaked at the beginning of the day and gradually decreased to trough levels at night. Recording of locomotor activity revealed higher daytime activity of C57Bl than of C3H. Our results indicate that the expression of Ectonucleotidases varies according to time and genotype and suggest that melatonin exerts modulatory effects associated with different regulations of purinergic signaling in the brain. These findings provide an important basis for further examination of the complexity of the purinergic system in the brain.
-
dysfunctional cd39 pos regulatory t cells and aberrant control of t helper type 17 cells in autoimmune hepatitis
Hepatology, 2014Co-Authors: Charlotte R Grant, Maria Serena Longhi, Simon C. Robson, Rodrigo Liberal, Beth Holder, John Cardone, Giorgina Mielivergani, D VerganiAbstract:Autoimmune hepatitis (AIH) is an important cause of severe liver disease and is associated with both quantitative and qualitative regulatory T-cell (Treg) impairments. Tregs express CD39, an Ectonucleotidase responsible for extracellular nucleotide hydrolysis, culminating in the production of immunosuppressive adenosine. Here, we describe multiple CD39pos Treg defects that potentially contribute to the impaired immunoregulation that is characteristic of AIH. We have examined the frequency and phenotype of CD39pos Tregs by flow cytometry and measured their Ectonucleotidase activity. The capacity of CD4posCD25high, CD4posCD25highCD39pos, and CD4posCD25highCD39neg subsets to suppress both proliferation of effector T cells and interleukin (IL)-17 production was evaluated. In AIH, CD39pos Tregs are decreased in frequency, exhibit limited adenosine triphosphate/adenosine diphosphate hydrolysis activity, and fail to suppress IL-17 production by effector CD4 T cells. Moreover, these CD39pos Tregs display a more proinflammatory profile in AIH, which is characterized by elevated CD127 positivity, and a greater propensity to produce interferon-gamma or IL-17 upon challenge with proinflammatory stimuli. Conclusions: In AIH, CD39pos Tregs are decreased in number, fail to adequately hydrolyze proinflammatory nucleotides and do not efficiently suppress IL-17 production by effector CD4 T cells. CD39pos Tregs show plasticity and are unstable upon proinflammatory challenge, suggesting that defective immunoregulation in AIH might result not only from reduced Treg number and function, but also from increased conversion of Tregs into effector cells. (Hepatology 2014;59:1007–1015)
-
vascular smooth muscle cell expression of Ectonucleotidase cd39 entpd1 is required for neointimal formation in mice
Purinergic Signalling, 2009Co-Authors: Amir Behdad, Keiichi Enjyoji, Xiaofeng Sun, Zain Khalpey, Marcia Wink, Anny Usheva, Simon C. RobsonAbstract:Vascular smooth muscle cell (VSMC) migration and proliferation are critical steps in the pathogenesis of atherosclerosis, post-angioplasty restenosis, neointimal hyperplasia, and chronic allograft rejection. Extracellular nucleotides are known to influence both migration and proliferation of VSMC. Although it is well established that vascular endothelial Cd39/ENTPD1 regulates blood nucleotide concentrations, whether Cd39 associated with VSMC also impacts vascular wall pathology has not been investigated. The objective of this paper is to determine levels of expression of Cd39 on VSMC and functional consequences of gene deletion in vitro and in vivo. Cd39 is the major Ectonucleotidase in VSMC, as shown by substantive decreases in ecto-ATPase and -ADPase activity in Cd39-null cells compared to wild type. Significant decreases in neointimal lesion formation are observed in Cd39-null mice at 21 days post arterial balloon injury. Stimulated Cd39-null VSMC have pronounced proliferative responses in vitro. However, using Transwell systems, we show that Cd39-null VSMC fail to migrate in response to ATP, UTP, and PDGF. Cd39 is the dominant Ectonucleotidase expressed by VSMC. Deletion of Cd39 in mice results in decreased neointimal formation after vascular injury and is associated with impaired VSMC migration responses in vitro.
-
natural killer t cell dysfunction in cd39 null mice protects against concanavalin a induced hepatitis
Hepatology, 2008Co-Authors: Guido Beldi, Keiichi Enjyoji, Lindsay Miller, Yara Banz, Michael Nowak, Arvand Haschemi, Gennady G Yegutkin, Daniel Candinas, Mark A Exley, Simon C. RobsonAbstract:Concanavalin A (Con A) administration results in murine liver injury that is thought to be mediated by T cells, natural killer T (NKT) cells, and antigen-presenting cells such as dendritic cells.1,2 Hepatic NKT cells are highly enriched in rodent liver3 and are required for Con A–induced hepatitis.4–6 CD1d-deficient mice that lack all NKT cells appear resistant to Con A–induced hepatitis.1,4–6 Similarly, sufficient numbers of NKT cells are required to propagate hepatic injury.7,8 Importantly, the activation of NKT cells is associated with a degree of apoptosis that occurs rapidly after induction by Con A or the NKT cell–specific ligand α-galactosylceramide (αGalCer).4,9,10 NKT cell–linked injury is associated with the secretion of the cytokines interleukin 4 (IL-4), interferon-γ (IFN-γ), and tumor necrosis factor-α.5,6,11,12 In addition to cytokines, extracellular nucleotides also accumulate at inflammatory sites. These latter mediators modulate immune reactions and are operative through the activation of specific P2Y and P2X receptors that are expressed on many cell types.13–17 Lymphocytes release adenosine triphosphate (ATP) and accumulate a halo of pericellular nucleotides upon stimulation with polyclonal stimuli such as anti-CD318,19 or mitogenic lectins such as Con A.20 Activation of these P2 surface receptors regulates lymphocyte and leukocyte functions such as cytokine secretion and/or migration.21,22 As a pertinent example, lymphoid cell activation and proliferation in response to Con A is closely associated with the activation of P2 receptors.23,24 Interestingly, mice deficient in the ATP receptor P2X7 are resistant to Con A–induced hepatitis.25 However, in such mice null for P2X7, the NKT cells exhibit a dimorphic phenotype in which responses appear to be dictated by the prior state of activation. Activation of P2X7 receptors on naive cells induces inhibitory signals, whereas in primed cells the activation responses appear to be facilitated further. It remains unclear whether putative alterations in levels of extracellular nucleotides that develop during hepatic injury might impact the inflammatory response in a NKT cell–dependent manner. In a closely related manner, adenosine, an end product of nucleotide hydrolysis, has potent anti-inflammatory and immune suppressive properties. These effects are executed by activation of certain P1 receptors. Anti-inflammatory outcomes have been demonstrated to involve the adenosine A2A receptor in various liver injury models, including Con A–induced hepatitis.26 Levels of extracellular nucleotides and the generation of adenosine are tightly regulated by cell surface ectoenzymes known as Ectonucleotidases. Within the vasculature, CD39 is crucial for the hydrolysis of extracellular nucleotides such as ATP and adenosine diphosphate (ADP) to the respective monophosphates, and, in concert with 5′ Ectonucleotidase or CD73, these generate adenosine. 27 To test whether the major Ectonucleotidase CD39 is expressed by NKT cells and alters the outcome of Con A–induced hepatitis, we have derived and tested mice deficient in CD39.28 We describe a novel phenotype of hepatic NKT cells in that these cells express both Ectonucleotidases that operate in tandem to regulate membrane P2 receptors. We also note how disordered metabolism of extracellular nucleotides following on genetic deletion of CD39 protects against immune liver injury in a murine model of Con A–induced hepatitis. We provide evidence that the mechanism involves heightened apoptosis of hepatic NKT cells in mutant mice resulting in hepatoprotection.
Jean Sevigny - One of the best experts on this subject based on the ideXlab platform.
-
sulfonylhydrazones design synthesis and investigation of Ectonucleotidase alp e5 nt inhibition activities
Bioorganic Chemistry, 2020Co-Authors: Hafiza Amna Younus, Julie Pelletier, Abdul Hameed, Abid Mahmood, Muhammad Siraj Khan, Muhammad Saeed, Farwa Batool, Asnuzilawati Asari, Habsah Mohamad, Jean SevignyAbstract:Abstract Medicinal importance of the sulfonylhydrazones is well-evident owing to their binding ability with zinc containing metalloenzymes. In the present study, we have synthesized different series of sulfonylhydrazones by using facile synthetic methods in good to excellent yield. All the successfully prepared sulfonylhydrazones were screened for Ectonucleotidase (ALP & e5′NT) inhibitory activity. Among the chromen-2-one scaffold based sulfonylhydrazones, the compounds 7 was found to be most potent inhibitor for h-TNAP (human tissue non-specific alkaline phosphatase) and h-IAP (human intestinal alkaline phosphatase) with IC50 values of 1.02 ± 0.13 and 0.32 ± 0.0 3 µM respectively, compared with levamisole (IC50 = 25.2 ± 1.90 µM for h-TNAP) and l -phenylalanine (IC50 = 100 ± 3.00 µM for h-IAP) as standards. Further, the chromen-2-one based molecule 5a showed excellent activity against h-ecto 5′-NT (human ecto-5′-nucleotidase) with IC50 value of 0.29 ± 0.004 µM compared to standard, sulfamic acid (IC50 = 42.1 ± 7.8 µM). However, among the series of phenyl ring based sulfonylhydrazones, compound 9d was found to be most potent against h-TNAP and h-IAP with IC50 values of 0.85 ± 0.08 and 0.52 ± 0.03 µM, respectively. Moreover, in silico studies were also carried to demonstrate their putative binding with the target enzymes. The potent compounds 5a, 7, and 9d against different Ectonucleotidases (h-ecto 5′-NT, h-TNAP, h-IAP) could potentially serve as lead for the development of new therapeutic agents.
-
Ectonucleotidase inhibitory and redox activity of imidazole based organic salts and ionic liquids
ChemMedChem, 2018Co-Authors: Veaceslav Boldescu, Joanna Lecka, Jean Sevigny, Natalia Sucman, Sidra Hassan, Jamshed Iqbal, Mariana Neamtu, Denis Prodius, Fliur MacaevAbstract:Cytotoxicity against cancer and normal cells, inhibition of Ectonucleotidase, and redox properties of a new group of imidazole-based organic salts and ionic liquids were studied. The tetrachloroferrate salt of a 1-methylimidazole derivative of salicylic aldehyde had most prominent inhibitory activity against Ectonucleotidase as well as a higher cytotoxicity against HeLa cells and lower cytotoxicity against BHK-21 cells than the reference compound carboplatin. The studied compounds exhibited a moderate level of antioxidant activity with better results for the salicylic aldehyde derivatives than for spiropyrans. Moreover, these compounds did not generate singlet oxygen.
-
development of a selective and highly sensitive fluorescence assay for nucleoside triphosphate diphosphohydrolase1 ntpdase1 cd39
Analyst, 2018Co-Authors: Sangyong Lee, Jean Sevigny, Xihuan Luo, Vigneshwaran Namasivayam, Jennifer Geiss, Salahuddin Mirza, Julie Pelletier, Holger Stephan, Christa E. MüllerAbstract:Ecto-nucleoside triphosphate diphosphohydrolase1 (NTPDase1, CD39) is a major Ectonucleotidase that hydrolyzes proinflammatory ATP via ADP to AMP, which is subsequently converted by ecto-5′-nucleotidase (CD73) to immunosuppressive adenosine. Activation of CD39 has potential for treating inflammatory diseases, while inhibition was suggested as a novel strategy for the immunotherapy of cancer. In the present study, we developed a selective and highly sensitive capillary electrophoresis (CE) assay using a novel fluorescent CD39 substrate, a fluorescein-labelled ATP (PSB-170621A) that is converted to its AMP derivative. To accelerate the assays, a two-directional (forward and reverse) CE system was implemented using 96-well plates, which is suitable for the screening of compound libraries (Z′-factor: 0.7). The detection limits for the forward and reverse operation were 11.7 and 2.00 pM, respectively, indicating a large enhancement in sensitivity as compared to previous methods (e.g. malachite-green assay: 1 000 000-fold, CE-UV assay: 500 000-fold, fluorescence polarization immunoassay: 12 500-fold). Enzyme kinetic studies at human CD39 revealed a Km value of 19.6 μM, and a kcat value of 119 × 10−3 s−1 for PSB-170621A, which shows similar substrate properties as ATP (11.4 μM and 82.5 × 10−3 s−1). The compound displayed similar properties at rat and mouse CD39. Subsequent docking studies into a homology model of human CD39 revealed a hydrophobic pocket that accommodates the fluorescein tag. PSB-170621A was found to be preferably hydrolyzed by CD39 as compared to other Ectonucleotidases. The new assay was validated by performing inhibition assays with several standard CD39 inhibitors yielding results that were consonant with data using the natural substrates.
-
coexpression of ecto 5 nucleotidase cd73 with specific ntpdases differentially regulates adenosine formation in the rat liver
American Journal of Physiology-gastrointestinal and Liver Physiology, 2012Co-Authors: Michel Fausther, Joanna Lecka, Julie Pelletier, Gilles Kauffenstein, Elwy Soliman, Nina Sheung, Jonathan A Dranoff, Jean SevignyAbstract:Ectonucleotidases modulate purinergic signaling by hydrolyzing ATP to adenosine. Here we characterized the impact of the cellular distribution of hepatic Ectonucleotidases, namely nucleoside triphosphate diphosphohydrolase (NTPDase)1/CD39, NTPDase2/CD39L1, NTPDase8, and ecto-5′-nucleotidase/CD73, and of their specific biochemical properties, on the levels of P1 and P2 receptor agonists, with an emphasis on adenosine-producing CD73. Immunostaining and enzyme histochemistry showed that the distribution of CD73 (protein and AMPase activity) overlaps partially with those of NTPDase1, -2, and -8 (protein levels and ATPase and ADPase activities) in normal rat liver. CD73 is expressed in fibroblastic cells located underneath vascular endothelial cells and smooth muscle cells, which both express NTPDase1, in portal spaces in a distinct fibroblast population next to NTPDase2-positive portal fibroblasts, and in bile canaliculi, together with NTPDase8. In fibrotic rat livers, CD73 protein expression and activity are redistributed but still overlap with the NTPDases mentioned. The ability of the observed combinations of Ectonucleotidases to generate adenosine over time was evaluated by reverse-phase HPLC with the recombinant rat enzymes at high “inflammatory” (500 μM) and low “physiological” (1 μM) ATP concentrations. Overall, ATP was rapidly converted to adenosine by the NTPDase1+CD73 combination, but not by the NTPDase2+CD73 combination. In the presence of NTPDase8 and CD73, ATP was sequentially dephosphorylated to the CD73 inhibitor ADP, and then to AMP, thus resulting in a delayed formation of adenosine. In conclusion, the specific cellular cocompartmentalization of CD73 with hepatic NTPDases is not redundant and may lead to the differential activation of P1 and P2 receptors, under normal and fibrotic conditions.
-
impact of ectoenzymes on p2 and p1 receptor signaling
Advances in pharmacology (San Diego), 2011Co-Authors: Filip Kukulski, Sebastien A Levesque, Jean SevignyAbstract:Abstract P2 receptors that are activated by extracellular nucleotides (e.g., ATP, ADP, UTP, UDP, Ap n A) and P1 receptors activated by adenosine control a diversity of biological processes. The activation of these receptors is tightly regulated by ectoenzymes that metabolize their ligands. This review presents these enzymes as well as their roles in the regulation of P2 and P1 receptor activation. We focus specifically on the role of ectoenzymes in processes of our interest, that is, inflammation, vascular tone, and neurotransmission. An update on the development of Ectonucleotidase inhibitors is also presented.
Ana Maria Oliveira Battastini - One of the best experts on this subject based on the ideXlab platform.
-
nucleoside triphosphate diphosphohydrolase 2 ntpdase2 cd39l1 is the dominant Ectonucleotidase expressed by rat astrocytes
Neuroscience, 2006Co-Authors: Marcia R Wink, Jean Sevigny, Elizandra Braganhol, Alessandra Sayuri Kikuchi Tamajusuku, Guido Lenz, Luiz F Zerbini, Towia A Libermann, Ana Maria Oliveira BattastiniAbstract:Inflammatory and degenerative pathophysiological processes within the CNS are important causes of human disease. Astrocytes appear to modulate these reactions and are a major source of inflammatory mediators, e.g. extracellular adenine nucleotides, in nervous tissues. Actions following extracellular nucleotides binding to type 2 purinergic receptors are regulated by Ectonucleotidases, including members of the CD39/ecto-nucleoside triphosphate diphosphohydrolase family. The Ectonucleotidases of astrocytes expressed by rat brain rapidly convert extracellular ATP to ADP, ultimately to AMP. RT-PCR, immunocytochemistry as well as Western blotting analysis demonstrated expression of multiple ecto-nucleoside triphosphate diphosphohydrolase family members at both the mRNA and protein level. By quantitative real-time PCR, we identified Entpd2 (CD39L1) as the dominant Entpd gene expressed by rat hippocampal, cortical and cerebellar astrocytes. These data in combination with the elevated ecto-ATPase activity observed in these brain regions, suggest that NTPDase2, an ecto-enzyme that preferentially hydrolyzes ATP, is the major ecto-nucleoside triphosphate diphosphohydrolase expressed by rat astrocytes. NTPDase2 may modulate inflammatory reactions within the CNS and could represent a useful therapeutic target in human disease.
-
effects of steroid hormones on synaptosomal Ectonucleotidase activities from hippocampus and cortex of adult female rats
General and Comparative Endocrinology, 2005Co-Authors: Barbara Rucker, Ana Maria Oliveira Battastini, Cristina Ribas Furstenau, Maria Luiza Morais Barretochaves, Daniela Pochmann, Marcela Sorelli Carneiroramos, João José Freitas SarkisAbstract:Abstract Over the last few years, the effects of steroid hormones on the brain have been intensively discussed. It has been demonstrated that ATP (acting as a neurotransmitter) is hydrolyzed to adenosine in the synaptic cleft by the conjugated action of Ectonucleotidases, which include an enzyme of the E-NTPDase family (NTPDase3, apyrase, EC 3.6.1.5) and a 5′-nucleotidase (EC 3.1.3.5). The 5′-nucleotidase enzyme is able to hydrolyze AMP as well as other monophosphate nucleotides. The importance of this enzyme in the central nervous system is to participate in the adenosine formation, a nucleoside with neuroprotective properties and modulatory effects. However, several questions have been raised about the mechanisms of steroid hormones and the possible neuroprotective effects of estrogen. Thus, we examined the effects of gonadal steroid hormone deprivation, induced by ovary removal (OVX) and estradiol replacement therapy, on the Ectonucleotidase activities in synaptosomes from hippocampus and cerebral cortex of adult rats. ATP and ADP hydrolysis in synaptosomes from cerebral cortex and hippocampus did not change as a function of OVX and results demonstrated an increase in AMP hydrolysis (82%) in the animals submitted to OVX in cerebral cortex, but not in hippocampus, when compared to control and sham-operated groups. Estradiol replacement therapy reversed this effect. RT-PCR analysis showed that the enhancement of enzyme activity in cerebral cortex could be explained by the higher expression of 5′-nucleotidase, following OVX. The hormones 17β-estradiol (cyclodextrin-encapsulated 17β-estradiol), DHEAS, and pregnenolone (1.0, 2.5, and 5.0 μM) did not alter the nucleotide hydrolysis, in vitro, in synaptosomes from cortex and hippocampus of female adult rats. Results presented, herein, should be considered relevant for hormone replacement therapy, since much controversy exists surrounding this area and the relationship between adenosine and sex steroids is still poorly understood.
-
changes in Ectonucleotidase activities in rat sertoli cells during sexual maturation
Molecular and Cellular Biochemistry, 2003Co-Authors: Emerson Andre Casali, Ana Maria Oliveira Battastini, Luiz Fernando De Souza, Daniel Pens Gelain, Gloria Regina Rodrigues De Freitas Kaiser, João José Freitas SarkisAbstract:Sertoli cell maturation is a complex process involving both morphological and biochemical changes. These cells have previously been shown to be targets for extracellular purine structures such as ATP and adenosine. These compounds evoke responses in rat Sertoli cells through the purinoceptor families, P2X and P2Y and PA1. The signals to purinoceptors are usually terminated by the action of Ectonucleotidases. In a previous work, we demonstrated that rat Sertoli cells have ecto-ATPdiphosphohydrolase (EC 3.6.1.5), ecto-5′-nucleotidase (EC 3.1.3.5) and ecto-adenosine deaminase (ecto-ADA) (EC 3.5.4.4) activities. Here we investigated whether some changes occur during rat Sertoli cell maturation in these activities. Rat Sertoli cells obtained from rats of different ages representing the pre pubertal, mid pubertal and ‘young adult’ (10-, 18- and 35-day-old, respectively) were cultured and used for different assays. The nucleotide hydrolysis was estimated by measuring the Pi released using a colorimetric method and by HPLC analysis. ATP and ADP hydrolysis was increased 3-fold during sexual maturation. AMP hydrolysis increased 4-fold in 10- to 35-day-old Sertoli cells. Similar results were obtained when we used other substrates to measure the extracellular hydrolysis of nucleotides (GTP, GDP, GMP and IMP). The ecto-ADA activity showed a 2-fold increase in the specific activity (18- to 35-day-old Sertoli cells). The termination of the purine cascade by adenosine degradation was faster in the 35- than in 18-day-old Sertoli cells. Follicle Stimulating Hormone (FSH) influences on the Ectonucleotidase activities were investigated in 10- and 18-day-old Sertoli cells and a significant increase in the ATP and ADP hydrolysis was observed. Our results show an increase in the extracellular purine cascade during the Sertoli cell development, indicating a rise in the purine communication inside the seminiferous tubules with rat sexual maturation.
-
in vitro effects of thyroid hormones on Ectonucleotidase activities in synaptosomes from hippocampus of rats
Cellular and Molecular Neurobiology, 2002Co-Authors: Jose Matos, Carla Denise Bonan, Ana Maria Oliveira Battastini, Maria Luiza Morais Barretochaves, Alessandra Nejar Bruno, Jean Pierre Oses, João José Freitas SarkisAbstract:1. Studies have shown that adenosine transport and adenosine A1 receptors in rat brain are subjected to regulation by thyroid hormone levels. Since the Ectonucleotidase pathway is an important source of adenosine extracellular, in the present study the in vitro action of T3 and T4 hormones on Ectonucleotidase activities in hippocampal synaptosomes was evaluated.
-
effects of inhibitory avoidance training and or isolated foot shock on Ectonucleotidase activities in synaptosomes of the anterior and posterior cingulate cortex and the medial precentral area of adult rats
Behavioural Brain Research, 2002Co-Authors: Grace S Pereira, João José Freitas Sarkis, Carla Denise Bonan, Ana Maria Oliveira Battastini, Ivan Izquierdo, Tadeu Mello E SouzaAbstract:Compelling evidence has indicated the involvement of extracellular ATP and adenosine in the mechanisms of synaptic plasticity and memory formation. In the present study, adult rats were trained in a step-down inhibitory avoidance task (IA) or submitted to isolated foot-shock (IF) (0.4 mA) before measuring Ectonucleotidase activities in the synaptosomes of the anterior and posterior cingulate cortex (AC and PC, respectively) and the medial precentral area (Fr2). IA increased ATP and ADP hydrolysis immediately after training in the synaptosomes of PC and AC, respectively, (P<0.05). Foot-shock (independent of occurring during IA or IF) increased ATP hydrolysis in synaptosomes of AC and Fr2 immediately after application and decreased AIP hydrolysis in AC 90 min after application (P<0.05). Foot-shock (independent of occurring during IA or IF) increased ATP hydrolysis in PC immediately and 90 min after application, and in Fr2, but only immediately after application (P<0.05). These results suggest that the Ectonucleotidase pathway responds to a mild foot-shock in AC, PC and Fr2 and may be involved in memory consolidation of step-down inhibitory avoidance in the cingulate cortex.
