Ectonucleoside Triphosphate Diphosphohydrolase Research Articles

Lambda-cyhalothrin exposure alters purine nucleotide hydrolysis and nucleotidase gene expression pattern in platelets and liver of rats

Lambda-cyhalothrin (LCT) is a broad-spectrum pesticide widely used in agriculture throughout the world. This pesticide is considered a potential contaminant of surface and underground water as well as food, posing a risk to ecosystems and humans. In this sense, we decided to evaluate the activity of enzymes belonging to the purinergic system, which is linked with regulation of extracellular nucleotides and nucleosides, such as adenosine triphosphate (ATP) and adenosine (Ado) molecules involved in the regulation of inflammatory response. However, there are no data concerning the effects of LCT exposure on the purinergic system, where extracellular nucleotides act as signaling molecules. The aim of this study was to evaluate nucleotide hydrolysis by E-NTPDase (ecto-nucleoside triphosphate diphosphohydrolase), Ecto-NPP (ecto-nucleotide pyrophosphatase/phosphodiesterase), ecto-5′-nucleotidase and ecto-adenosine deaminase (E-ADA) in platelets and liver of adult rats on days 7, 30, 45 and 60 after daily gavage with 6.2 and 31.1 mg/kg bw of LCT. Gene expression patterns of NTPDases1–3 and 5′-nucleotidase were also determined in those tissues. In parallel, lambda-cyhalothrin metabolites [3-(2-chloro-3,3,3- trifluoroprop-1-enyl)-2,2-dimethyl-cyclopropane carboxylic acid (CFMP), 4-hydroxyphenoxybenzoic acid (4-OH-3-PBA), and 3-phenoxybenzoic acid (3-PBA)] were measured in plasma. Results showed that exposure rats to LCT caused a significant increase in the assessed enzymes activities. Gene expression pattern of ectonucleotidases further revealed a significant increase in E-NTPDase1, E-NTPDase2, and E-NTPDase3 mRNA levels after LCT administration at all times. A dose-dependent increase in LCT metabolite levels was also observed but there no significant variations in levels from weeks to week, suggesting steady-steady equilibrium. Correlation analyses revealed that LCT metabolites in the liver and plasma were positively correlated with the adenine nucleotides hydrolyzing enzyme, E-ADA and E-NPP activities in platelets and liver of rats exposed to lambda-cyhalothin.Our results show that LCT and its metabolites may affect purinergic enzymatic cascade and cause alterations in energy metabolism.

CD73-dependent adenosine dampens interleukin-1β-induced CXCL8 production in gingival fibroblasts: Association with heme oxygenase-1 and adenosine monophosphate-activated protein kinase.

During inflammation, stressed or infected cells can release adenosine triphosphate (ATP) to the extracellular medium, which can be hydrolyzed to adenosine by ectonucleotidases such as ectonucleoside triphosphate diphosphohydrolase 1 (CD39) and 5'-nucleotidase (CD73). The role of CD73 in the modulation of cytokine release by human gingival fibroblasts (HGFs) remains underexplored. Here, we investigated whether CD73-mediated hydrolysis of extracellular ATP (eATP) could affect interleukin (IL)-1β-induced CXCL8 secretion. The levels of mRNA expression of adenosine receptors, CD39 and CD73 of periodontitis samples were retrieved from a public database. Moreover, HGF mRNA levels were measured by quantitative reverse transcription-polymerase chain reaction (RT-qPCR) after 3, 6, or 24hours of IL-1β stimulation. IL-1β-induced CXCL8 protein levels were measured after pretreatment with 100-µM eATP in the presence or absence of CD73 inhibitor. The effect of eATP degradation to adenosine on CXCL8 levels was investigated using agonist and antagonist of adenosine receptors. Levels of CD39, CD73, and adenosine receptor mRNA were differentially modulated by IL-1β. ATP pretreatment impaired IL-1β-induced CXCL8 secretion and required activation of heme oxygenase-1 (HO-1) and phosphorylated adenosine monophosphate-activated protein kinase (pAMPK). The inhibition of CD73 or the inhibition of adenosine receptors abrogated the ATP effect on CXCL8 secretion. CD73-generated adenosine dampens IL-1β-induced CXCL8 in HGFs and involves HO-1 and pAMPK signaling. These results imply that CD73 is a negative regulator of the inflammatory microenvironment, suggesting that this ectoenzyme could be involved in the generation of deficient CXCL8 gradient in chronic inflammation.

Effect of quercetin on E-NTPDase/E-ADA activities and cytokine secretion of complete Freund adjuvant-induced arthritic rats.

The effect of quercetin was assessed in rats induced with complete Freund adjuvant (CFA). Arthritis scores, paw oedema, latency, activities of myeloperoxidase (MPO), ectonucleoside triphosphate diphosphohydrolase (E-NTPDase), and ectoadenosine deaminase (E-ADA) in lymphocytes were determined. Furthermore, nucleotide and nucleoside levels as well as the secretion of pro- and anti-inflammatory cytokines were evaluated. Animals were treated with saline and quercetin in doses of 5, 25, and 50mg/kg for 45days. The result revealed that quercetin (50mg/kg) reduced arthritis score and paw oedema, and increased the latency in the thermal hyperalgesia test. Histopathological analysis showed that all the doses of quercetin reduced infiltration of inflammatory cells. MPO activity was increased in the arthritis group; however, quercetin reduced this activity. E-NTPDase activity was increased in lymphocytes of arthritis rats, and treatment with quercetin reversed this increase. However, E-ADA activity was reduced in the arthritis group, and treatment with quercetin modulated the activity of this enzyme in arthritis rat groups. Serum adenosine levels were increased in arthritis, and the levels were lowered with quercetin treatment. Quercetin treatment in arthritis groups decreased the elevated levels of cytokines in the arthritis control group. Thus, quercetin demonstrated an anti-inflammatory effect, and this flavonoid may be a promising natural compound for the treatment of arthritis. SIGNIFICANCE OF THE STUDY: Quercetin may represent a potential therapeutic compound in the treatment of rheumatoid arthritis. Findings from this study indicate that quercetin suppresses swelling and attenuates the underlying inflammatory responses. This is the first report where quercetin was shown to modulate the immune response to arthritis via attenuation of the purinergic system (E-NTPDase and E-ADA activities) and the levels of IFN-gamma and IL-4. Thus, this work is relevant to basic research and may be translated into clinical practice.

Purinergic Signaling in Pulmonary Inflammation.

Purine nucleotides and nucleosides are at the center of biologic reactions. In particular, adenosine triphosphate (ATP) is the fundamental energy currency of cellular activity and adenosine has been demonstrated to play essential roles in human physiology and pathophysiology. In this review, we examine the role of purinergic signaling in acute and chronic pulmonary inflammation, with emphasis on ATP and adenosine. ATP is released into extracellular space in response to cellular injury and necrosis. It is then metabolized to adenosine monophosphate (AMP) via ectonucleoside triphosphate diphosphohydrolase-1 (CD39) and further hydrolyzed to adenosine via ecto-5′-nucleotidase (CD73). Adenosine signals via one of four adenosine receptors to exert pro- or anti-inflammatory effects. Adenosine signaling is terminated by intracellular transport by concentrative or equilibrative nucleoside transporters (CNTs and ENTs), deamination to inosine by adenosine deaminase (ADA), or phosphorylation back into AMP via adenosine kinase (AK). Pulmonary inflammatory and hypoxic conditions lead to increased extracellular ATP, adenosine diphosphate (ADP) and adenosine levels, which translates to increased adenosine signaling. Adenosine signaling is central to the pulmonary injury response, leading to various effects on inflammation, repair and remodeling processes that are either tissue-protective or tissue destructive. In the acute setting, particularly through activation of adenosine 2A and 2B receptors, adenosine signaling serves an anti-inflammatory, tissue-protective role. However, excessive adenosine signaling in the chronic setting promotes pro-inflammatory, tissue destructive effects in chronic pulmonary inflammation.

Identifying small molecule probes of ENTPD5 through high throughput screening.

Ectonucleoside Triphosphate Diphosphohydrolase 5 (ENTPD5) has been shown to be important in maintaining cellular function in cancer, and its expression is upregulated through multiple, unique pathways in certain cancers, including laryngeal, glioblastoma multiforme, breast, testicular, and prostate. ENTPD5 supports cancer growth by promoting the import of UDP-glucose, a metabolite used for protein glycosylation and hence proper glycoprotein folding, into the ER by providing the counter molecule, UMP, to the ER antiporter. Despite its cancer-supporting function, no small molecule inhibitors of ENTPD5 are commercially available, and few studies have been performed in tissue culture to understand the effects of chemical inhibition of ENTPD5. We performed a high-throughput screen (HTS) of 21,120 compounds to identify small molecule inhibitors of ENPTD5 activity. Two hits were identified, and we performed a structure activity relationship (SAR) screen around these hits. Further validation of these probes were done in an orthogonal assay and then assayed in cell culture to assess their effect on prostate cancer cell lines. Notably, treatment with the novel ENTPD5 inhibitor reduced the amount of glycoprotein produced in treated cells, consistent with the hypothesis that ENTPD5 is important for glycoprotein folding. This work serves as an important step in designing new molecular probes for ENTPD5 as well as further probing the utility of targeting ENTPD5 to combat cancer cell proliferation.

Eggplant (Solanum spp) supplemented fruits diet modulated the activities of ectonucleoside triphosphate diphosphohydrolase (ENTPdase), monoamine oxidase (MAO), and cholinesterases (AChE/BChE) in the brain of diabetic Wistar male rats.

Type 2 diabetes mellitus is associated with complications such as Alzheimer disease (AD). Tropical eggplant (Solanum gilo, Solanum kumba, and Solanum aethiopicum) fruits have been extensively used for the treatment of different ailments. This study assesses the effect of an eggplant supplemented-diet on purinergic, monoaminergic, and cholinergic enzyme systems in diabetic male rats, besides determining the presence of alkaloids using GC-MS chromatography. Results from this study show that eggplant fruit diet modulates the activities of the enzymes in purinergic, monoaminergic, and cholinergic enzyme systems associated with AD-like symptoms. Solanum kumba-supplemented diet significantly (p<0.05) reduced enzyme activities better than S. gilo and S. aethiopicum, which could be due to its rich phytochemical constituents. In conclusion, eggplant fruits could serve as a holistic measure in the prevention of diabetes-related complications such as neurodegenerative disease. PRACTICAL APPLICATIONS: The therapeutic management of diabetes fails to holistically address inflammatory response which likely contributes to type 2 diabetes mellitus (T2DM) occurrence by causing insulin resistance; this, in turn, is intensified in the presence of hyperglycemia to promote long-term complications such as neurodegenerative disorders. The health benefit of a tropical eggplant fruit diet inform a nutritional and therapeutic approach for the prevention and treatment of T2DM and its associated complications such as neurodegenerative disorders has been proved. The eggplant fruit-supplemented diet, which is cost-effective with little or no side effect, could substantially increase the antioxidant status and also modulate the activities of neuronal enzymes in a diabetic model with dementia, as well as Alzheimer's-like symptoms. This study, therefore, revealed more of the benefits of tropical eggplant fruits vis-à-vis their management in hyperglycemia-mediated neurodegeneration.

Proteomic study of pig’s spleen

This work is devoted to pig spleen proteome study. Spleens were taken from Duroc pigs (females, 145 - 160 days old) and typical two-dimensional electrophoregrams were obtained. On proteomic maps after visualization and image analysis there were detected 600 fractions, including organ-specific proteins - 3 62 fractions. Among the identified constitutive fractions, the highest expression was observed (Vol spots more than 3.0E + 07) four protein spots S1, S9, S12 and S21, which are supposedly Annexin A1 (MW 38.76 kDa), Ectonucleoside triphosphate diphosphohydrolase 1 (MW 57.75 kDa) Pro-cathepsin H CD59 (MW 37.45 kDa) and glycoprotein (MW 13.79 kDa), respectively. Obtained electrophoregrams analysis using information resources made it possible to identify different active compounds in spleen with various functions, mainly immunoregulatory - glycoprotein CD59 (Mm 13.79 kDa) and ATP-dependent RNA helicase (Mm 107.58 kDa); the intensely expressed LIM-domain of the actin-binding protein (Mm 83.99 kDa). The results obtained are a prospect for immunomodulating biologic development based on animal raw materials for farm animals.

Identification and characterization of ecto-nucleoside triphosphate diphosphohydrolase 1 (CD39) involved in regulating extracellular ATP-mediated innate immune responses in Japanese flounder (Paralichthys olivaceus)

Extracellular adenosine triphosphate (eATP), released following inflammatory stimulation or infection, is a potent signaling molecule in activating innate immune responses in fish. However, the regulation of eATP-mediated innate immunity in fish remains unknown. Ecto-nucleoside triphosphate diphosphohydrolase 1 (CD39) is a critical molecular switch for controlling the ATP levels in the extracellular space. CD39 plays a key role in regulating eATP-activated innate immune responses through the phosphohydrolysis of pro-inflammatory eATP to inactive AMP. Here, we identified and characterized a CD39 homolog (namely, poCD39) in the Japanese flounder Paralichthys olivaceus and analyzed its regulatory role in eATP-mediated innate immunity. Real-time quantitative PCR analysis revealed that poCD39 is ubiquitously present in all tested normal tissues with dominant expression in enriched Japanese flounder head kidney macrophages (HKMs). Immune challenge experiments demonstrated that poCD39 expression was upregulated by inflammatory stimulation and Edwardsiella tarda infection. Biochemical and immunofluorescence analysis revealed that poCD39 is a functional glycosylated membrane protein for the hydrolysis of eATP. Inhibition of poCD939 activity with the ecto-NTPDase inhibitor ARL 67156 resulted in increased IL-1beta gene expression and ROS production in Japanese flounder HKMs. In contrast, overexpression of poCD39 in Japanese flounder FG-9307 cells reduced eATP-induced pro-inflammatory cytokine IL-1beta gene expression. Finally, poCD39 expression was significantly induced by eATP stimulation in the HKMs, suggesting that eATP may provide a feedback mechanism for transcriptional regulation of fish CD39. Taken together, we identified and characterized a functional fish CD39 protein involved in regulating eATP-mediated innate immune responses in fish.

CD133+ bone marrow stem cells (BMSC) control platelet activation – Role of ectoNTPDase-1 (CD39)

BackgroundWe previously demonstrated CD133+ bone marrow stem cells (BMSC) to promote hepatic proliferation for liver regeneration. Here, we evaluated the capacity of CD133+BMSC to utilize platelets for homing to vasculature and concomitant controlling their aggregability upon ADP stimulation. MethodsCD133+BMSC and platelets were co-cultured along micro endothelial cells under variable flow conditions and tested for homing levels along vasculature. Aggregometry and FACS analysis were utilized to evaluate platelet reactivity following co-incubation ± CD133+BMSC. RT-PCR and FACS analyses served to characterize ADP degrading ectonucleoside triphosphate diphosphohydrolase-1 (ectoNTPDase-1/CD39) expression on various cell types. ResultsPlatelets attracted human CD133+BMSC to autologous micro endothelium under shear stress unaffected by ADP stimulation. However, CD133+BMSC inhibited ADP-mediated platelet activation and aggregation. Latter was dependent on ectoNTPDase-1 expression levels. Platelet aggregatory control was increased with CD133+BMSC compared to CD133+PHSC. Different effects of those stem cell subtypes positively correlated with their FACS-detected expression levels of ectoNTPDase-1. ConclusionWe provide evidence that CD133+BMSC are capable of controlling ADP-dependent platelet aggregation and activation by direct interaction dependent on cellular expression of ectoNTPDase-1. Whether different capacities of BMSC modulate platelet-depending thrombogenicity at sites of regeneration impact effectiveness and adverse event profiles of regenerative treatment requires further evaluation.

Induction of NTPDase1/CD39 by Reactive Microglia and Macrophages Is Associated With the Functional State During EAE.

Purinergic signaling is critically involved in neuroinflammation associated with multiple sclerosis (MS) and its major inflammatory animal model, experimental autoimmune encephalomyelitis (EAE). Herein, we explored the expression of ectonucleoside triphosphate diphosphohydrolase1 (NTPDase1/CD39) in the spinal cord, at the onset (Eo), peak (Ep), and end (Ee) of EAE. Several-fold increase in mRNA and in NTPDase1 protein levels were observed at Eo and Ep. In situ hybridization combined with fluorescent immunohistochemistry showed that reactive microglia and infiltrated mononuclear cells mostly accounted for the observed increase. Colocalization analysis revealed that up to 80% of Iba1 immunoreactivity and ∼50% of CD68 immunoreactivity was colocalized with NTPDase1, while flow cytometric analysis revealed that ∼70% of mononuclear infiltrates were NTPDase1+ at Ep. Given the main role of NTPDase1 to degrade proinflammatory ATP, we hypothesized that the observed up-regulation of NTPDase1 may be associated with the transition between proinflammatory M1-like to neuroprotective M2-like phenotype of microglia/macrophages during EAE. Functional phenotype of reactive microglia/macrophages that overexpress NTPDase1 was assessed by multi-image colocalization analysis using iNOS and Arg1 as selective markers for M1 and M2 reactive states, respectively. At the peak of EAE NTPDase1 immunoreactivity showed much higher co-occurrence with Arg1 immunoreactivity in microglia and macrophages, compared to iNOS, implying its stronger association with M2-like reactive phenotype. Additionally, in ∼80% of CD68 positive cells NTPDase1 was coexpressed with Arg1 compared to negligible fraction coexpresing iNOS and ∼15% coexpresing both markers, additionally indicating prevalent association of NTPDase1 with M2-like microglial/macrophages phenotype at Ep. Together, our data suggest an association between NTPDase1 up-regulation by reactive microglia and infiltrated macrophages and their transition toward antiinflammatory phenotype in EAE.

Effects of Endogenous ω6:ω3 PUFA Ratio Reduction on Ileum Homeostasis and Liver Injury in Mice Chronically Fed Ethanol

The objective of the current study was to test the hypothesis that increasing endogenous levels of w3 PUFAs alters gene expression profiles in the intestine of mice chronically fed an ethanol (EtOH) diet, leading to improved gut barrier and liver function, in a mouse model of alcoholic liver disease (ALD). ALD is a significant human health issue leading to increased morbibity and mortality resulting from liver dysfunction. Chronic alcohol consumption causes changes in intestinal permeability resulting in translocation of the gut luminal contents into the circulation. Therefore, a complete understanding of the mechanism(s) by which alcohol consumption alters gut barrier function is essential in order to develop therapeutic strategies to combat these effects. To test the above hypothesis, wild‐type and fat‐1 transgenic mice (which express an w6 fatty acid desaturase and therefore can directly convert w6 to w3 PUFAs) were fed a liquid diet with or without EtOH (pair‐fed, PF) for 6 weeks. Prior to euthanasia, a subset of the mice were challenged with LPS, which induces endotoxemia and is a frequent comorbidity to ALD. At the end of the study, liver and intestine pathology was evaluated and an RNA‐seq analysis was performed on ileum tissue. We identified numerous, statistically significant changes in ileum gene expression in fat‐1 mice when compared to wild‐type controls. Significant changes in the expression of genes that encode enzymes important for the metabolism and detoxification of EtOH were found in both pair‐fed and EtOH‐fed, LPS‐challenged mice. For example, the expression of Adh1 and Adh6a, two members of the alcohol dehydrogenase family, were significantly increased in the ileums of fat‐1 mice. In addition, several isoforms of aldehyde dehydrogenase were increased in fat‐1 mice, including Aldh1a7 and Aldh3b1, in both pair‐fed and EtOH/LPS‐treated animals. The expression of catalase, which metabolizes hydrogen peroxide, was increased and the expression of Cyp2e1 was decreased in PF fat‐1 mice, suggesting that these mice are better positioned to resist tissue damage as a result of reactive oxygen species. In mice fed only EtOH and not challenged with LPS, there was a significant change in the expression of genes important for xenobiotic metabolism. These include several cytochrome p450s (Cyp2c65, Cyp2d10, and Cyp2d34). Lastly, we found that the expression many genes involved in the metabolism of purines was significantly altered in the ileums of fat‐1 mice. For example, Entpd8 (ectonucleoside triphosphate diphosphohydrolase 8), Enpp3 (ectonucleotide pyrophosphatase/phosphodiesterase 3) and Ada (adenosine deaminase) were all increased in fat‐1 mice. The metabolism of adenosine is critically important for regulating immune function and therefore, altered purine metabolism may be one mechanism by which fat‐1 mice are protected from the toxic effects of chronic EtOH consumption. In summary, these data suggest that fat‐1 mice are much more efficient at metabolizing EtOH and the toxic metabolite, acetaldehyde, and have changes in purine metabolism that potentially have a beneficial effect on overall intestine function.Support or Funding InformationNational Institutes of HealthThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A new case of spastic paraplegia type 64 due to a missense mutation in the ENTPD1 gene

Spastic paraplegia type 64 (SPG64; OMIM 615683) is a complicated form of hereditary spastic paraplegia (HSP) recently identified in individuals diagnosed with suspected neurodegenerative disease. Affected patients carry homozygous mutations in the ectonucleoside triphosphate diphosphohydrolase 1 gene (ENTPD1). Although they share common characteristics, affected individuals show slight discrepancies in some clinical aspects. At present, only two different cases of SPG64 have been diagnosed. More findings of genetic variation would be helpful to better understand the effect of mutations in the ENTPD1 gene on the neurological condition of affected individuals. In this study, we examined a family with an individual diagnosed with suspected HSP based on clinical findings. DNA samples from the proband, her affected sister, and both parents were analyzed using next-generation sequencing. We used an in-house automated pipeline to detect potential neuromuscular disease-causing variants. Variants were confirmed by Sanger sequencing. After cosegregation analysis, the variant NM_001776.5:c.401T>G (p.M134R) of the ENTPD1 gene was identified as a novel missense mutation linked to the phenotype of SPG64 in the proband and her sister, who showed similar and distinct clinical features compared with the two cases previously described in the literature.

GATA Binding Protein 3 Boosts Extracellular ATP Hydrolysis and Inhibits Metastasis of Breast Cancer by Up-regulating Ectonucleoside Triphosphate Diphosphohydrolase 3.

Despite remarkable advancements in our understanding of breast cancer, it remains the leading cause of cancer deaths in women. Distant recurrence and metastasis is the main reason for death due to breast cancer. It is well recognized that the GATA binding protein 3 (GATA3), a transcription factor, is a tumor suppressor in breast cancer. To date, the mechanistic molecular details of GATA3 remain elusive, because, as a transcription factor, it is not a direct executor in physiological and pathological processes. Here, we demonstrate that GATA3 reduces the ATP level in the breast cancer microenvironment and inhibits breast cancer metastasis by up-regulating ectonucleoside triphosphate diphosphohydrolase 3 (ENTPD3). The extracellular ATP concentration is significantly higher in tumor tissues than in normal tissues and promotes the migration of cancer cells from the primary site. ENTPD3 hydrolyzes ATP in tumor microenvironment and suppresses breast cancer metastasis. Furthermore, ENTPD3 inhibits epithelial-to-mesenchymal transition, a key program responsible for the development of metastatic disease. These findings provide novel insights into the tumor suppressor activity of GATA3.

Role of Ectonucleotidases in Synapse Formation During Brain Development: Physiological and Pathological Implications.

Background Extracellular adenine nucleotides and nucleosides, such as ATP and adenosine, are among the most recently identified and least investigated diffusible signaling factors that contribute to the structural and functional remodeling of the brain, both during embryonic and postnatal development. Their levels in the extracellular milieu are tightly controlled by various ectonucleotidases: ecto-nucleotide pyrophosphatase/phosphodiesterases (E-NPP), alkaline phosphatases (AP), ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) and ecto-5'-nucleotidase (eN).Methods Studies related to the expression patterns of ectonucleotidases and their known features during brain development are reviewed, highlighting involvement of these enzymes in synapse formation and maturation in physiological as well as in pathological states.Results During brain development and in adulthood all ectonucleotidases have diverse expression pattern, cell specific localization and function. NPPs are expressed at early embryonic days, but the expression of NPP3 is reduced and restricted to ependymal area in adult brain. NTPDase2 is dominant ectonucleotidase existing in the progenitor cells as well as main astrocytic NTPDase in the adult brain, while NTPDase3 is fully expressed after third postnatal week, almost exclusively on varicose fibers. Specific brain AP is functionally associated with synapse formation and this enzyme is sufficient for adenosine production during neurite growth and peak of synaptogenesis. eN is transiently associated with synapses during synaptogenesis, however in adult brain it is more glial than neuronal enzyme.Conclusion Control of extracellular adenine nucleotide levels by ectonucleotidases are important for understanding the role of purinergic signaling in developing tissues and potential targets in developmental disorders such as autism.

Extracellular ATP and CD39 Regulates Mitochondrial Function and Cytarabine Resistance through Intrinsic PKA-ATF-PGC1a Pathway in Acute Myeloid Leukemia

Relapses in acute myeloid leukemia (AML) are caused by chemoresistant leukemic populations and new therapeutic approaches that specifically target these cells are urgently needed. Based on transcriptomic analyses of relevant PDX preclinical model of the resistance to cytarabine (AraC) and of the residual disease in patients, we identified ecto-nucleoside triphosphate diphosphohydrolase-1 CD39 (ENTPD1) overexpressed in the residual leukemic cells in vivo after chemotherapy. By flow cytometry, we confirmed that AraC increased cell surface CD39 expression in AML cell lines in vitro and in vivo as well as in 24 diverse patient-derived xenograft models. We further observed this increase in 100 patients at 35-days post-intensive chemotherapy compared to their respective diagnosis. Interestingly, high CD39 expression on AML patients was associated with a worse response to AraC in vivo. Furthermore, we showed that FACS-sorted CD39high AML cells had increased mitochondrial mass and activity, and were resistant to AraC in vitro and in vivo. We demonstrated that CD39 downstream signaling pathway was dependent on cAMP-PKA-PGC1a-TFAM axis and its inhibition by H89 sensitized AML cells to AraC through the inhibition of mitochondrial OxPHOS biogenesis and function. Finally, pharmacological inhibition of CD39 ATP hydrolase activity or genetic invalidation of CD39 protein using two inhibitors or shRNA markedly enhanced AraC cytotoxicity in AML cell lines and primary patient samples in vitro and in vivo. Together, these results indicate CD39 as a new player of the intrinsic chemoresistance pathway and a new therapeutic target to specifically overcome AraC resistance and eradicate these leukemic cells responsible for relapses in AML. DisclosuresNo relevant conflicts of interest to declare.

Neuroprotective effects of berberine on recognition memory impairment, oxidative stress, and damage to the purinergic system in rats submitted to intracerebroventricular injection of streptozotocin.

Alzheimer's disease (AD) is a progressive and irreversible neurodegenerative disease. The present study investigated the effects of 50 and 100mg/kg berberine (BRB) on recognition memory, oxidative stress, and purinergic neurotransmission, in a model of sporadic dementia of the Alzheimer's type induced by intracerebroventricular (ICV) injection of streptozotocin (STZ) in rats. Rats were submitted to ICV-STZ 3mg/kg or saline, and 3days later, were started on a treatment of BRB or saline for 21days. The results demonstrated that BRB was effective in protecting against memory impairment, increased reactive oxygen species, and the subsequent increase in protein and lipid oxidation in the cerebral cortex and hippocampus, as well as δ-aminolevulinate dehydratase inhibition in the cerebral cortex. Moreover, the decrease in total thiols, and the reduced glutathione and glutathione S-transferase activity in the cerebral cortex and hippocampus of ICV-STZ rats, was prevented by BRB treatment. Besides an antioxidant effect, BRB treatment was capable of preventing decreases in ecto-nucleoside triphosphate diphosphohydrolase (NTPDase), 5'-nucleotidase (EC-5'-Nt), and adenosine deaminase (ADA) activities in synaptosomes of the cerebral cortex and hippocampus. Thus, our data suggest that BRB exerts a neuroprotective effect on recognition memory, as well as on oxidative stress and oxidative stress-related damage, such as dysfunction of the purinergic system. This suggests that BRB may act as a promising multipotent agent for the treatment of AD.

Activity and expression of E-NTPDase is altered in peripheral lymphocytes of systemic lupus erythematosus patients

BackgroundSystemic lupus erythematosus (SLE) is an inflammatory autoimmune disease, where there is irreversible breakdown of immunological self-tolerance. Extracellular adenosine triphosphate (ATP) and adenosine are signaling molecules that play an important part in the immune response. During inflammation and the immune response, a group of enzymes control these molecules, including ectonucleoside triphosphate diphosphohydrolase (E-NTPDase), E-5′-nucleotidase, and ecto-adenosine deaminase (E-ADA). We determined the activity and expression of E-NTPDase, the expression of E-5′-nucleotidase, the activity of E-ADA in lymphocytes and serum of SLE patients. MethodsThis study involved 35 patients with SLE and 30 healthy subjects as a control group. E-NTPDase activity and expression were increased in lymphocytes from SLE patients (31% and 37% for activity and expression, respectively) compared with the control group. ResultsAn approximately 42% increase in E-ADA activity in lymphocytes was observed in SLE patients compared with the control group, in serum the ADA activity was decreased by 57% in SLE patients. Expression of E-5′-nucleotidase was not changed in SLE patients. ConclusionsE-NTPDase and E-ADA perform key functions in the modulation of the immune and inflammatory response in SLE.

Synthesis of Extracellular Pyrophosphate Increases in Vascular Smooth Muscle Cells During Phosphate-Induced Calcification.

Objective- Hydroxyapatite deposition on the medial layer of the aortic walls is the hallmark of vascular calcification and the most common complication in aging individuals and in patients with diabetes mellitus and those undergoing hemodialysis. Extracellular pyrophosphate is a potent physicochemical inhibitor of hydroxyapatite crystal formation. This study analyzed changes in extracellular pyrophosphate metabolism during the phosphate-induced calcification process. Approach and Results- Phosphate-induced calcification of ex vivo-cultured aortic rings resulted in calcium accumulation after 7 days. This accumulation was enhanced when aortic walls were devitalized. BMP2 (bone morphogenic protein 2) expression was associated with calcium accumulation in cultured aortic rings, as well as in cultured vascular smooth muscle cells (VSMCs) and in calcitriol-induced calcification in rats. Hydroxyapatite dose dependently induced BMP2 overexpression in VSMCs. Moreover, TNAP (tissue nonspecific alkaline phosphatase) mRNA levels and activity were found to be downregulated in early phases and upregulated in later phases of calcification in all 3 models studied. eNPP1 (ectonucleotide pyrophosphatase/phosphodiesterase 1) increased from early to later phases of calcification, whereas eNTPD1 (ectonucleoside triphosphate diphosphohydrolase 1) was downregulated during later phases. Synthesis of pyrophosphate in VSMCs increased significantly over time, in all 3 models studied. Because the rate of pyrophosphate hydrolysis was 10× slower than the rate of pyrophosphate synthesis, pyrophosphate synthesis is determined mainly by the ratio of eNPP1 to eNTPD1 activity. Hydroxyapatite also induces increments both in TNAP and eNPP1/eNTPD1 ratio in VSMCs. Conclusions- Pyrophosphate synthesis increases in VSMCs during phosphate-induced calcification because of compensatory regulation of extracellular pyrophosphate metabolism.

Purinergic signalling displays a pro-inflammatory profile in spleen and splenic lymphocytes of Rhamdia quelen fed with a diet contaminated by fungal mycotoxin: Involvement on disease pathogenesis

The spleen is an important secondary lymphatic organ that plays a key role in the immune and inflammatory responses of teleost fish. The purinergic signalling has been associated to these types of responses under pathological conditions by the regulation of extracellular adenosine triphosphate (ATP) and its metabolite adenosine (Ado), where both exert potent pro-inflammatory and anti-inflammatory profiles, respectively. The exact pathway involved on the immunotoxic effects of aflatoxin B1 (AFB1) in fish fed with diets containing this mycotoxin remains poorly understood. Thus, the aim of this study was to evaluate whether purinergic signalling exerts anti or pro-inflammatory effects in spleen and splenic lymphocytes of Rhamdia quelen fed with a diet contaminated by AFB1. Ectonucleoside triphosphate diphosphohydrolase (NTPDase) activity (ATP as substrate) decreased in spleen and splenic lymphocytes of fish fed with an AFB1-contaminated diet on day 21 post-feeding compared to fish fed with a basal diet; while adenosine deaminase (ADA) activity increased. No differences were observed between groups or over time regarding NTPDase (adenosine diphosphate as substrate) and 5′-nucleotidase activities. In summary, the purinergic signalling can be a pathway involved in the impairment of the immune and inflammatory responses in fish fed with an AFB1-contaminated diet, contributing to the immunotoxic effects of AFB1 in spleens of fish.

Purinergic system as a potential target for inflammation and toxicity induced by thymol in immune cells and tissues

Thymol is a phytochemical component present in many plants used as food additive in order to promote animal growth due to its several biological properties. However, possible side effects of thymol remain poorly known limited to few reports. In this sense, we evaluated the enzymes of the purinergic signaling such as, ectonucleoside triphosphate diphosphohydrolase (NTPDase), 5'-nucleotidase and adenosine deaminase (ADA), that play an important role on toxicity induced by excessive adenosine triphosphate (ATP) content in the extracellular environment. Thus, the aim of this study was to evaluate whether purinergic signaling could be considered a potential target of thymol-induced inflammation, and the toxicity in tissues and immune cells of mice after thymol administration. NTPDase activity (ATP as substrate) in serum, spleen, and splenic lymphocytes was lower after 30 days of oral treatment at doses of 10, 20, and 40mg/kg of thymol, while ADA activity was stimulated at 20 and 40mg/kg. No differences were observed between groups regarding NTPDase (ADP as substrate) and 5'-nucleotidase activities in all evaluated tissues. Based on these evidences, adenine nucleotide hydrolysis is modified in serum, spleen, and splenic lymphocytes of mice treated with thymol, contributing to inflammation and toxicity by a reduction on ATP hydrolyses and its possible accumulation in the extracellular medium and increased Ado desamination and its possible reduction in the extracellular environment, leading to a self-sustained pro-inflammatory deleterious cycle. In summary, all tested thymol concentrations induced inflammation and toxicity in tissues and immune cells of treated mice.